Tumorigenesis is known to result from multiple genetic changes, which are often manifested as gross chromosome abnormalities. These can involve a single chromosomal translocation or be manifested as massive genome-wide instability. Although endogenous and environmental insults can damage DNA, robust cellular mechanisms exist to repair various forms of damage or to eliminate those cells that have been irreparably damaged. Hence, the accumulation of numerous genetic changes that would lead to cancer in normal cells is extremely rare. Nevertheless, disruption of a DNA repair pathway has the potential to expedite tumorigenesis by resulting in a cell that is hypermutable. DNA damage in the form of chromosomal double-strand breaks (DSBs) occurs spontaneously as a result of normal DNA metabolism and after exposure to exogenous DNA damaging agents, such as ionizing radiation. We demonstrated that a major pathway involved in DSB repair is homologous recombination in which an unbroken sequence templates the repair of a broken sequence to which it is homologous (i.e., homology-directed repair, or HDR). Other major DSB repair pathways are non-homologous end-joining (NHEJ), in which broken ends are rejoined using little or no sequence homology, and single-strand annealing. Recently, it has become clear that HDR and NHEJ mutants exhibit chromosome instability, both spontaneously and damage induced. Our aim is to begin to understand the contribution of different repair pathways in normal cells to DSB repair, and how this is altered in DSB repair mutants. In Specific Aim 1, the contributions of DSB repair pathways in mouse embryonic stem cells will be examined and compared with repair in fibroblasts, as the reliance on repair factors for IR sensitivity varies in these cell types. In Specific Aim 2, we will determine if an NHEJ mutant demonstrates a genetic dependence on a factor involved in HDR by constructing double mutants of the Rad54 and Ku80 genes, which are involved in HDR and NHEJ, respectively. In Specific Aim 3, the effect of NHEJ mutations on molecular mechanisms of chromosomal translocations will be examined. We will exploit a system we have recently developed to induce chromosomal translocations and examine the effect of NHEJ mutations on the frequency and outcomes of these events. In Specific Aim 4, we will examine the dynamics of DSB repair. We have thus far focused on the outcome of DSB repair after I-Scel cleavage of a chromosome. In this aim we will develop the DSB induction system to understand the dynamics of DSB repair in terms of the proteins associated with the repair process, the relative kinetics of different DSB repair processes, and initial processing steps at a DSB.